DOI: 10.1126/scitranslmed.3009759, 263ra158 (2014);6 Sci Transl Med

et al.Viorica BranisteThe gut microbiota influences blood-brain barrier permeability in mice

 Editor's Summary

   

the gut microbiota and the brain, initiated during the intrauterine period, is perpetuated throughout life.permeability, possibly through the regulation of tight junction proteins. These findings suggest that crosstalk between mice. However, fecal transplants from mice exposed to bacteria into adult germ-free mice reduced blood-brain barrierincreased permeability of the blood-brain barrier. This elevated permeability was also observed in adult germ-free

now show that germ-free pregnant dams, devoid of maternal microbes, have offspring that showet al.Braniste brain.of the brain. An intact blood-brain barrier is a crucial checkpoint for appropriate development and function of the

The blood-brain barrier is an important gateway that controls the passage of molecules and nutrients in and out

The Gut Microbiota and the Blood-Brain Barrier

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R E S EARCH ART I C L E

BLOOD -BRA IN BARR I ER

The gut microbiota influences blood-brain barrierpermeability in miceViorica Braniste,1*† Maha Al-Asmakh,1* Czeslawa Kowal,2* Farhana Anuar,1 Afrouz Abbaspour,1

Miklós Tóth,3 Agata Korecka,1 Nadja Bakocevic,4 Ng Lai Guan,4 Parag Kundu,5 Balázs Gulyás,3,5

Christer Halldin,3,5 Kjell Hultenby,6 Harriet Nilsson,7 Hans Hebert,7 Bruce T. Volpe,8

Betty Diamond,2‡ Sven Pettersson1,5,9†‡

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Pivotal to brain development and function is an intact blood-brain barrier (BBB), which acts as a gatekeeper tocontrol the passage and exchange of molecules and nutrients between the circulatory system and the brainparenchyma. The BBB also ensures homeostasis of the central nervous system (CNS). We report that germ-freemice, beginning with intrauterine life, displayed increased BBB permeability compared to pathogen-free micewith a normal gut flora. The increased BBB permeability was maintained in germ-free mice after birth andduring adulthood and was associated with reduced expression of the tight junction proteins occludin and claudin-5,which are known to regulate barrier function in endothelial tissues. Exposure of germ-free adult mice to apathogen-free gut microbiota decreased BBB permeability and up-regulated the expression of tight junctionproteins. Our results suggest that gut microbiota–BBB communication is initiated during gestation and propa-gated throughout life.

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INTRODUCTION

Our gut microbiota is important for many biological functions in thebody, including intestinal development, barrier integrity and function(1, 2), metabolism (3, 4), the immune system (5), and the central ner-vous system (CNS). The effects of the gut microbiota on brain phys-iology include synaptogenesis, regulation of neurotransmitters andneurotrophic factors such as brain-derived neurotrophic factor andnerve growth factor-A1 (6). However, the development of the CNSalso includes the formation of an intact blood-brain barrier (BBB) thatensures an optimal microenvironment for neuronal growth and spec-ification (7). An intact and tightly regulated BBB is also required toprotect against colonizing microbiota in neonates during the criticalperiod of brain development (8, 9). It also protects against exposure to“new”molecules and bacterial metabolites due to the postnatal metabolicswitch from predominant dependence on carbohydrates during fetallife to a greater dependence on fatty acid catabolism after birth.

The BBB begins to develop during the early period of intrauterinelife (10, 11) and is formed by capillary endothelial cells sealed by tightjunctions, astrocytes, and pericytes. Tight junction proteins restrictingparacellular diffusion of water-soluble substances from blood to thebrain (12) consist mainly of transmembrane proteins such as claudins,

1Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, 17177 Stockholm,Sweden. 2Center for Autoimmune andMusculoskeletal Disease, The Feinstein Institute forMedical Research, North Shore-LIJ Health System, Manhasset, NY 11030, USA. 3PsychiatrySection, Department of Clinical Neuroscience, Karolinska Institutet, 17176 Stockholm,Sweden. 4Singapore Immunology Network, Agency for Science, Technology and Research,Singapore 138648, Singapore. 5Lee Kong Chian School of Medicine, Nanyang Technolog-ical University, 60 Nanyang Drive, Singapore 637551, Singapore. 6Department of LaboratoryMedicine, Karolinska Institutet, 14186 Stockholm, Sweden. 7Department of Biosciencesand Nutrition, Karolinska Institutet, and School of Technology and Health, KTH Royal In-stitute of Technology, Novum, SE-141 57 Huddinge, Sweden. 8Laboratory of FunctionalNeuroanatomy, The Feinstein Institute for Medical Research, North Shore-LIJ Health System,Manhasset, NY 11030, USA. 9Singapore Centre on Environmental Life Sciences Engineering(SCELSE), Nanyang Technological University, Singapore 637551, Singapore.*Co-first authors.†Corresponding author. E-mail: viorica.braniste@ki.se (V.B.); sven.pettersson@ki.se (S.P.)‡Co-senior authors.

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tricellulin, and occludin, which are connected to the actin cytoskeletonby the zona occludens (ZO-1) (13). Tight junction proteins are dynamicstructures and are subject to changes in expression, subcellular location,posttranslational modification, and protein-protein interactions underboth physiological and pathophysiological conditions (12). Disruptionof tight junctions due to disease or drugs can lead to impaired BBBfunction, compromising the CNS. Therefore, understanding how BBBtight junction proteins are affected by various factors is important forelucidating how to prevent and treat neurological diseases.

Here,we report that the intestinalmicrobiota affects BBBpermeabilityin both the fetal and adult mouse brain. Using as a model system germ-freemice that have never encountered a live bacteriumandpathogen-freemice that were reared in an environment free ofmonitoredmouse patho-gens, we demonstrated that lack of gut microbiota is associated withincreased BBB permeability and altered expression of tight junction pro-teins. Fecal transfer frommicewithpathogen-free gut flora into germ-freemice or treatment of germ-free mice with bacteria that produce shortchain fatty acids (SCFA) decreased the permeability of the BBB.

RESULTS

The maternal gut microbiota can influence prenataldevelopment of the BBBFirst, we characterized BBB permeability of mouse embryos with pathogen-free mothers by administering infrared-labeled immunoglobulinG2b (IgG2b) antibody to dams during timed pregnancies to see wheth-er the antibody was excluded by the BBB or was able to cross the BBBinto the brain parenchyma. The qualitative analysis of mouse embryoswith pathogen-free mothers showed a shift from a diffuse infrared-labeledantibody signal present within the embryonic brain at E13.5 and E14.5to a signal confined to the developing vasculature starting at E15.5 toE17.5 (Fig. 1A). This signal was most pronounced in adult offspring ofpathogen-free dams (Fig. 1A). The quantitative analysis of the pene-tration into the fetal brain of infrared-labeled IgG2b antibody injected

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intravenously into pathogen-free dams supported the qualitative data,showing a decrease at E15.5 to E17.5 (Fig. 1B). In contrast, the analysisof E16.5 brains from fetal mice of germ-free dams showed a diffuse sig-nal from the infrared-labeled IgG2b antibody (Fig. 1C) present in the

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brain parenchyma (Fig. 1, D and E). Higher-magnification images of the brain showedthat the IgG2b antibody was limited only tothe vessels in E16.5 fetal mice of pathogen-free dams in contrast to age-matched fetalmice of germ-free dams (Fig. 1, D and E).Because BBB integrity is controlled in partby sealing of the endothelial cells via tightjunctions, we determined expression of themain tight junction proteins in brain ly-sates from E18.5 fetal mice of pathogen-free versus germ-free dams. Expression ofthe brain endothelial tight junction proteinsclaudin-5 and ZO-1 was similar betweenthe two groups, whereas the expression ofoccludin was significantly lower in the brainlysates from E18.5 fetal mice of germ-freedams compared to that in age-matchedfetal mice of pathogen-free dams (P < 0.05)(Fig. 1, F and G).

Lack of gut microbiota isassociated with increased BBBpermeability in adult miceThree techniques were used to determinewhether the BBB was more permeable ingerm-free adult mice: (i) in vivo positronemission tomography (PET) imaging with[11C]raclopride (Fig. 2, A to C); (ii) extra-vasation of Evans blue tracer from thecirculation (Fig. 2D); and (iii) the capacityof an anti–N-methyl-D-aspartate receptorreactive antibody (R4A) to induce neuro-nal death after intravenous administration(Fig. 2, E and F).

In germ-free adult mice, [11C]racloprideuptake was increased compared with thatfor pathogen-free adult mice (Fig. 2A), butonly during the first 4 min after injection(Fig. 2, B and C). This period of time rep-resents the “flow phase” (that is, the pres-ence of the radioligand in the whole braindue to BBB permeability). Because the ra-dioligand was given in tracer doses, it doesnot exert any pharmacological effects onthe brain (or body) vasculature or heartrate. These differences were present onlyin the initial flow phase and not in the laterphase of the time activity curves, indicatingno differences in radioligand binding todopamine D2 receptors between the groups.

Fluorescence microscopy images of dif-ferent brain regions (cortex, striatum, andhippocampus) of pathogen-free adult mice

showed the presence of Evans blue dye (bright red) only in the blood vessels,whereas Evans blue staining in germ-free mice was detected not only in theblood vessels but also in the brain parenchyma, demonstrating leakage of thedye across the BBB (Fig. 2D). A group of mice receiving an intravenous

Fig. 1. BBB integrity in fetal mice with germ- or pathogen-free mothers. (A) Representative lateralimages of the brains of E13.5 to E17.5 mouse embryos and adult mice (ventral) 1 hour after infrared-

labeled antibody was injected into pregnant pathogen-free (PF) mothers. Scale bar, 1 mm. (B) Quantitativeanalysis of antibody penetration into the fetal brain of mice with pathogen-free mothers. Data are ex-pressed as means ± SEM (7 to 12 embryos per group). ***P < 0.0001 between E17.5 group versus therest of the groups by one-way analysis of variance (ANOVA). (C) Representative images from the infrared-labeled antibody assay in E16.5 mouse embryos with germ-free (GF) mothers. Scale bar, 1 mm. (D) Sagittalbrain sections fromeach of three E16.5mouse embryoswith germ- or pathogen-freemothers after injectingthe dam with IgG. IgG (top row of each pair, Alexa 594), CD31 [platelet endothelial cell adhesion molecule(PECAM); bottom row of each pair, Alexa 488]. Scale bar, 500 mm. (E) Maternal IgG in comparable regions ofthe brain of E16.5 mouse embryos. Left column: IgG (Alexa 594). Middle column: CD31 (PECAM; Alexa 488).Right column: Merged images. Scale bar, 20 mm. (F and G) Western blots of brain lysates from E18.5 mousefetuses with germ- or pathogen-free mothers probed for ZO-1, occludin, claudin-5, and glyceraldehydephosphate dehydrogenase (GAPDH) (control). (F) Representative blots and (G) quantification. Black bars,PF. White bars, GF. Datawere normalized for GAPDH expression and expressed as fold change, control fold(c.f.) PF. Data are means ± SEM (four to six mice per group). *P < 0.05 by Student’s t test. ns, not significant.

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injection of tumor necrosis factor–a (TNFa) 15 hours before the ex-periment served as a positive control for BBB leakiness (Fig. 2D).

In germ-free adult mice, intravenous administration of the mono-clonal antibody R4A (250 mg) was associated with abnormal neurons,marked by condensed cytoplasm and shrunken cell bodies in the CA1region of the hippocampus (Fig. 2E, right panel). Abnormal neuronswere not present or were rare in the CA1 region of the hippocampusin the control group [phosphate-buffered saline (PBS)–treated germ-

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free group; Fig. 2E, left panel]. Furthermore, the R4A injection (at lowand high doses) in germ-free adult mice was associated with a signif-icant reduction in neuron numbers in the CA1 region of the hippo-campus, –38% for low-dose R4A (100 mg) and –42% for high-dose(250 mg) R4A compared with the PBS-treated germ-free control group(P < 0.01) (Fig. 2F). Intravenous administration of R4A in pathogen-free adult mice did not induce any changes, indicating that the R4Amonoclonal antibody did not penetrate the BBB (Fig. 2F).

Fig. 2. Increased BBB permeability in adult germ-free versus pathogen-free mice. (A) In vivo PET imaging of [11C]raclopride. Average coronal, sag-

and pathogen-freemice and pathogen-freemice treated with TNFa (n = 3miceper group). Blue, 4′,6-diamidino-2-phenylindole (DAPI) (nuclear staining). Scale

ittal, and horizontal PET summation images (brain area encircled in purpleellipse) in pathogen-free (PF) or germ-free (GF) adult mice 2 to 3 min after[11C]raclopride injection. (B) Average whole-brain time-activity curves of [11C]raclopride uptake expressed as % standardized uptake value (% SUV) in thetwo groups. *P < 0.05 and **P < 0.05 by one-way ANOVA. (C) Values (% SUV)obtained at 1-min intervals during the first 5 min. Data are expressed asmeans ± SEM (five to six mice per group). (D) Representative imagesshowing Evans blue dye extravasation (red) in three brain regions (cortex -upper row, striatum - middle row, and hippocampus - lower row) of germ-

bar, 50 mm. (E and F) Neuronal loss in the hippocampus of germ-free adultmice receiving R4A antibody. (E) Photomicrographs of the CA1 region of thehippocampus (middorsal, matched sections) from PBS-treated germ-free adultmice (left) and germ-free mice treated with different concentrations of R4Aantibody (100 and 250 mg) (right). Yellow arrows in the right panel indicateneuronal loss and dying neurons in R4A-treated germ-free mice. Scale bar,10 mm. (F) Quantitative analysis. Percent neuronal survival in the PBS-treatedgerm-free mouse group was set at 100%. **P < 0.01 by one-way ANOVA com-pared with the PBS-treated germ-free mouse group (n = 3 mice per group).

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Vascular density and pericyte coverage show no differencein germ- and pathogen-free adult miceWe used intravital two-photon microscopy to exclude the possibilitythat high BBB permeability in germ-free adult mice was caused byhigher vascular density in the brain. Using the second harmonic gen-eration signals from collagen fibers of dura mater as a reference point,we observed that the subdural region, which is 20 to 80 mm below thedura mater, contains mainly large vessels (average diameter, ~40 mm)(Fig. 3, A and C). In contrast, deeper regions of the brain 120 to 180 mmbelow the dura mater consist mostly of capillaries (Fig. 3, B and D).Quantitative analysis of vasculature density in the brain revealed no sig-nificant gross differences between germ- and pathogen-free adult mice(Fig. 3, C and D).

Pericytes play an important role in regulating BBB properties, anddecreased pericyte coverage has been associated with increased BBBpermeability (10, 14). Immunofluorescence staining using CD13, a cellsurface marker for pericytes in different brain regions, revealed nodifference in pericyte coverage between germ- and pathogen-free mice(Fig. 3E).

Brain endothelial tight junctions are altered in the absenceof a gut microbiotaPermeability of CNS vessels is controlled in part by dynamic openingand closing of the endothelial junctions (15). Therefore, we assessed

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the expression of the main tight junction proteins (ZO-1, occludin,and claudin-5) by Western blot in three regions of an adult mousebrain: frontal cortex, striatum, and hippocampus. Significantly lowerexpression of occludin and claudin-5 was observed in male germ-freemice compared with male pathogen-free mice in all three brain re-gions of interest (occludin: P < 0.001 in frontal cortex and hippocam-pus and P < 0.05 in striatum; claudin-5: P > 0.001 in frontal cortex andP < 0.01 in striatum and hippocampus) (Fig. 4, A to F). In contrast, nodifference in the expression of the cytoplasmic protein ZO-1 was ob-served between the two groups (Fig. 4, A to F). Similar patterns oftight junction protein expression were observed in the brains of femalegerm-free mice compared with female pathogen-free mice (fig. S1),suggesting that the effect of gut microbiota on the integrity of theBBB is independent of sex. In addition, immunofluorescence analysisconfirmed lower expression of occludin and claudin-5 in the brainvessels of adult male germ-free mice compared with that of adult malepathogen-free mice (Fig. 4, G and H).

The ultrastructure of the tight junctions was investigated by trans-mission electron microscopy analysis. In germ-free adult mice, thetight junctions appeared as a diffuse, disorganized structure comparedwith those in the pathogen-free group (Fig. 4I). A scoring system wasused to quantitatively determine the number of intact tight junctionsas follows: perfect tight junctions, 3; patches of blurriness, 2; totallyblurred, 1 (fig. S2 shows examples of the rating scale). In the striatum

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of germ-free adult mice, the number of in-tact tight junctions was significantly lowerthan that in pathogen-free mice (P <0.001) (Fig. 4J).

BBB permeability and tightjunction protein expression areassociated with changes inthe gut microbiotaColonization of germ-free adult mice withflora from pathogen-free mice [conventio-nalized (CONV)] was associated with in-creased integrity of the BBB as shown byrestriction of the Evans blue tracer to theblood vessels and decreased extravasationof the dye into the brain parenchyma(Fig. 5A). Quantitative analysis of tightjunction proteins in the CONV group com-pared with germ-free mice showed a signif-icant increase in the expression of occludinin the frontal cortex (P < 0.05) and stria-tum (P = 0.05) and of claudin-5 in the hip-pocampus and striatum (P < 0.05) (Fig. 5,B to G). Increased expression of the in-tracellular protein ZO-1 was detected inthe striatum and hippocampus of CONVmice compared with germ-free controls(P < 0.05) (Fig. 5, B to G).

SCFAs or metabolites produced bybacteria affect BBB permeabilitySCFAs are known to enhance the integrityof the intestinal epithelial barrier (16, 17)by facilitating the assembly of tight junctions

Fig. 3. Brain blood vessel and pericyte coverage in germ-and pathogen-free adult mice. (A to D) Two-photon imag-ing of brain blood vessels in germ-free (GF) and pathogen-free(PF) adult mice. Tetramethyl rhodamine isothiocyanate(TRITC)–dextran was applied retro-orbitally to highlight the brain

blood vessels. (A) Representative images of brain vasculature 20 to 80 mm below the dura mater in germ-free (left panel) and pathogen-free (right panel) mice reveal mainly large vessels (average diameter, ~40 mm).(B) Representative images of the brain vasculature 120 to 180 mm below the dura mater in germ-free (leftpanel) and pathogen-free (right panel) mice showing mainly capillaries. (C) Quantitative analysis of bloodvessel density 20 to 80 mm below the dura mater. (D) Quantitative analysis of blood vessel density 120 to180 mm below the dura mater. Scale bars, 100 mm. Data are representative of n = 3 independentexperiments. (E) Representative images of pericyte coverage (CD13, green) in the cerebral cortex ofpathogen- and germ-free mice (n = 4 mice per group). Laminin (red) was used as an endothelial cellmarker. Scale bars, 50 mm.

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(18). Hence, we evaluated BBB permeability in germ-free adult micemonocolonized with a single bacterial strain, Clostridium tyrobutyricum(CBut), that produces mainly butyrate (19, 20) or with Bacteroidesthetaiotaomicron (BTeta), which produces mainly acetate and propionate(21, 22). We also evaluated germ-free adult mice given sodium butyrate

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by oral gavage for 3 days. Evans blue perfusion in CBut-, BTeta-, andsodium butyrate–treated mice demonstrated decreased BBB per-meability, compared to that in germ-free adult mice, that was equivalentto that of pathogen-free adult mice (Fig. 6A). Administration of sodiumbutyrate to germ-free mice was associated with increased expression of

old change,s representper group).**P < 0.001ed with thefree mousentative im--free moused for endo-anti-laminin,nti–claudin-20 mm. (I)ing the dis-tructure be-in striatum

of germ-freestriatum ofpanel). (J)a decreasedt junctionsmice com-ice. Values

seven micey Student’s

Fig. 4. Disrupted BBB

tight junctions in thebrains of germ- andpathogen-free adultmice. (A to C) Rep-resentativeWestern blotsshowing the expressionof ZO-1, occludin, andclaudin-5 in the (A) fron-tal cortex, (B) striatum,and (C) hippocampusof germ-free (GF) andpathogen-free (PF) adultmice. (D to F) Densito-metric analysis of West-ern blots from proteinsamples of the (D) fron-tal cortex, (E) striatum,and (F) hippocampus ofgerm-free mice (whitebars) compared withpathogen-freemice (blackbars). Data were nor-malized for GAPDH ex- pression and expressed as fcontrol fold (c.f.) PF. Valuemeans ± SEM (6 to 10 mice*P < 0.05, **P < 0.01, and *by Student’s t test comparcorresponding pathogen-control. (G and H) Represeages of germ- and pathogencerebral motor cortex stainethelial cells with (G and H)(G) anti-occludin, and (H) a5 antibodies. Scale bars,Electron micrographs showorganized tight junction stween two endothelial cells(right panel, white arrows)adult mice compared withpathogen-free mice (leftQuantitative data indicatenumber of organized tighin the striatum of germ-freepared with pathogen-free mrepresent means ± SEM (per group). ***P < 0.001 bt test.

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occludin in the frontal cortex and hippocampus but had no effecton the expression of claudin-5 (Fig. 6, B to D). Furthermore, admin-istration of sodium butyrate or monocolonization of germ-free mice withC. tyrobutyricum was associated with an increase in histone acetylationin brain lysates (fig. S3).

DISCUSSION

The BBB is a physiological barrier that controls the passage of mole-cules between the brain parenchyma and the blood and in so doingallows proper functioning of neurons. Our results highlight the gut mi-crobiota as a potential regulator of BBB integrity. Here, we show thatthe lack of a normal gut microbiota in germ-free mice is associatedwith increased permeability of the BBB. This result was confirmedusing three different techniques: in vivo PET imaging using radio-labeled ligand, vascular leakage of Evans blue dye, and neuronal damage

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after intravenous administration of R4Aantibody. Furthermore, our data show thata more permeable BBB is observed in thefetal mice with germ-free mothers at E16.5to E18.5 days of embryonic developmentcompared to the fetal mice with pathogen-free mothers at the same stages of devel-opment. The increased permeability of theBBB in germ-free adult mice may partlybe the consequence of disorganized tightjunctions, as shown by electron microsco-py analysis, and low expression of thetransmembrane tight junction proteinsoccludin and claudin-5. The “conventionali-zation” of germ-free adult mice throughtransplant of the fecal microbiota frompathogen-free adult mice or by adminis-tering bacterial strains that produce SCFAsreinforced the integrity of the mouse BBB.

The BBB matures progressively dur-ing intrauterine life and continues to ma-ture during the early postnatal stages oflife (23). Our data confirm previous obser-vations (10, 24) and show that closure ofthe BBB to IgG in pathogen-free miceoccurs during the later stages of intra-uterine life. A recent study of Mfsd2a knock-out mice (lacking the transporter for theessential omega-3 fatty acid docosahexaenoicacid) showed that the BBB in mice be-comes functional at E15.5, demonstratingcomplex regional and temporal differencesin maturation (11). This coincides withour observation of the permeability of theembryonic BBB to maternal antibodies.In mice, gestational stage E15.5 is a turn-ing point for the restriction of maternalantibody penetration into the fetal brain.Maternal antibodies or, more precisely,antibody delivered to the embryo throughthe placenta was our molecule of choice

in our embryonic BBB studies as a physiological route of delivery. Re-duced closure of the BBB was observed in fetuses from germ-freedams. In humans, marked changes in the composition of the maternalgut microbiota have been observed between the first and the third tri-mesters of pregnancy (25). These observations, together with the presentstudy, imply that the maternal gut microbiota might contribute toincreased nutritional demands in late pregnancy, which would requiremore stringent control of BBB permeability in the growing offspring.

The BBB is a complex structure formed by capillary endothelial cells,pericytes, and astrocytes (26). A difference in vascular density mightbe a confounding factor in assessing BBB permeability. In our study,increased invasion of circulating substances into the brain parenchymaappears not to be due to differences in large vascular structures betweenthe two groups of mice as shown by a comparable equal visualizationof the brain vasculature using 140 kD TRITC-dextran. However, wecannot formally exclude that some microcapillary changes may stillexist between the two groups. In germ-free adult mice, pericyte coverage

Fig. 5. Microbial colonization of the gut changes BBB integrity. (A) Representative images from threemouse brain regions (frontal cortex, striatum, and hippocampus) showing Evans blue dye (red) in pathogen-

free (PF)mice, germ-free (GF)mice, andgerm-freemice colonizedwith pathogen-free flora for 14days (CONV).Blue, DAPI (nuclear staining). Scale bars, 50 mm. (B to G) Quantitative analysis of ZO-1, occludin, and claudin-5expression in the frontal cortex, striatum, and hippocampus of germ-free and CONVmice. Data were normal-ized for GAPDH expression and expressed as fold change, fold control (c.f.) GF. Values represent means ±SEM (four to six mice per group). *P < 0.05 by Student’s t test compared to the germ-free control.

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Fig. 6. The effect of SCFAs on BBB permeability. (A) Extravasation of Evansblue dye (red) observed in the brain regions (frontal cortex, striatum, and hip-

brain parenchyma. Blue, DAPI (nuclear staining). Scale bars, 50 mm. (B to D)Quantitative analysis of ZO-1, occludin, and claudin-5 expression in brain lysates

pocampus) of germ-free (GF) mice. In germ-free mice monocolonized witheither C. tyrobutyricum (CBut) or B. thetaiotaomicron (BTeta) for 2 weeks or micetreated with the bacterial metabolite sodiumbutyrate (NaBu) for 72 hours, Evansblue dye was detected only in the blood vessels, without any leakage into the

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from germ-free mice gavaged with water (GF) or NaBu for 72 hours. Data werenormalized for b-tubulin expression as a loading control and expressed as foldchange, control fold (c.f.) GF. Values are expressed as means ± SEM (four to fivemice per group). *P < 0.05 by Student’s t test compared to the germ-free control.

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was similar to that in pathogen-free mice, suggesting that altering thenumber of pericytes is unlikely to account for the increased BBB per-meability observed in germ-free mice. This is similar to the Mfsd2a-deficient mouse model in which leaky vessels are not associated withchanges in the cerebrovascular network or pericyte abnormalities (11).However, in contrast to the Mfsd2a-deficient mouse where transcytosisacross the BBB is affected without disruption of tight junctions, ourmodel implies that the gut microbiota may regulate the BBB throughmodulation of tight junction protein expression.

Tight junctions play a major role in the functional maintenance ofthe BBB (27, 28). Our data show disorganized tight junctions in thebrains of germ-free adult mice compared with those of pathogen-freemice, which was associated with lower expression of occludin and claudin-5.No difference in the expression of ZO-1 was observed. A decrease inoccludin and claudin-5 paralleled by cytoskeletal changes and tightjunction protein redistribution was associated with altered integrity ofthe BBB (29). Reduced expression of occludin was observed in germ-freemice during both intrauterine life and adulthood andwas associatedwithincreased permeability of the BBB.Administration of normal flora frompathogen-free mice or oral treatment with the bacterial metabolite so-diumbutyrate to germ-free adultmice induced an increase in the expres-sion of occludin that was associated with decreased permeability of theBBB. These observations imply that the expression of occludin by thebrain endothelial cells is sensitive to changes in the intestinal gutmicrobiota.

Regulation of occludin expression by the intestinal microbiota hasbeen reported in the intestinal epithelial barrier (30) and blood-testisbarrier (31). The expression of claudin-5 by brain endothelial cells waslow in germ-free adult mice but elevated after exposure to the gut mi-crobiota. However, no difference in the expression of claudin-5 was ob-served in the E18.5 cortex of fetal mice with germ- or pathogen-freemothers. This implies that claudin-5 expression continues to increaseduring the postnatal period in offspring of pathogen-free mothers butnot in offspring of germ-free mice. Although claudin-5 expression ispresent from early intrauterine life in the human brain, it is localizedmostly in the endothelial cytoplasm and shifts toward the endothelialborder during development to form tight junctions (32, 33). Further-more, claudin-5 knockout mice appear to develop normally duringintrauterine life with morphologically normal blood vessels but havea BBB impairment associated with increased permeability to smallmolecules (34). The complete depletion of claudin-5 is associated with100% mortality of the newborns within 10 hours of birth (34), furthersupporting the role of claudin-5 in the postnatal regulation of the BBB.

Dietary carbohydrates are substrates for fermentation by certain gutbacteria, which produce SCFAs, primarily acetate, propionate, andbutyrate, as end products (35). These SCFAs have been shown to reg-ulate intestinal motility (36, 37) and to be involved in central appetiteregulation (38, 39) as well as being taken up directly into the blood-stream and transported to various organs, including the brain (39, 40),where they modulate tissue development and function (41, 42). Phys-iological concentrations of SCFA mixtures or individual SCFAs regu-late intestinal barrier function by increasing the transepithelial electricalresistance and decreasing paracellular permeability (43). In a rat modelof transient focal cerebral ischemia, intraperitoneally injected sodiumbutyrate attenuated BBB disruption (6). In our study, we show thatmonocolonization of the intestine of germ-free adult mice with eitherC. tyrobutyricum, a bacterial strain producing butyrate (19, 44), orB. thetaiotaomicron, which produces mainly acetate and propionate (21),decreased BBB permeability. Oral administration of the bacterial me-

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tabolite sodium butyrate mimicked this effect on the BBB. The effectsof the other metabolites, acetate and propionate, as single substratesmay also have an effect on the permeability of the BBB and shouldalso be explored. Intravenous or intraperitoneal administration of so-dium butyrate has been reported to inhibit histone deacetylation andfacilitate long-term memory consolidation (45), prevent BBB break-down (46), and promote angiogenesis and neurogenesis (47, 48).Whether the gut microbiota and sodium butyrate alter histone acetyla-tion of brain microvascular endothelial cells requires further study tobetter understand the effects of SCFAs produced by the gut microbiotaon the CNS. Cumulative and chronic exposure to SCFAs could lead torelatively stable effects on gene expression in the brain.

Finally, the composition and diversity of the gut microbiota com-munity change over time, presumably reflecting different gut microbiota–host interactions. Early in life, there is an urgent need to support thegrowing offspring with an almost unlimited amount of energy to ensurebrain development. Germ-free mice show an increase in glucocorticoidproduction due to metabolic stress (49). An increase in BBB permeabil-ity may, therefore, allow serum glucocorticoids to enter the growingbrain, affecting neurogenesis and impairing production of brain-derivednerve growth factor in the brain (6). In addition, microbes regulatingthe BBB in infants with a growing brain may influence BBB perme-ability differently compared to the adult BBB (50).

Although providing a first glimpse into an additional layer of gut-brain communication, the current study has not revealed the precisesignaling mechanisms through which gut microbiota modulates BBBfunction. In addition, the consequences of increased BBB permeabilityin germ-free mice on neuronal function throughout life are still notknown. Therefore, the results should be interpreted cautiously untilmore physiological data are acquired to corroborate these findings.

MATERIALS AND METHODS

Study designThe objective of this study was to assess the importance of the intes-tinal microbiota in the maintenance of BBB integrity in a mouse mod-el. The integrity of the BBB was examined in germ-free mice and inpathogen-free mice using functional permeability assays and by deter-mining the status of tight junctions. BBB integrity was also determinedin a group of germ-free adult mice colonized with fecal samples frompathogen-free adult mice or treated with bacterial strains that producedSCFAs or the bacterial metabolite sodium butyrate.

AnimalsGerm- and pathogen-free NMRI (Naval Medical Research Institute)malemice andC57BL/6J female andmalemice (8 to 10weeks old) (CoreFacility forGerm-free Research, Karolinska Institutet) were used. C57BL/6J, Balb/c, and NMRI germ- and pathogen-free female mice undergoingtimed pregnancies were used to assess the intrauterine development of theBBB.Germ-freemicewere raised in special plastic isolators.All animalsweremaintained on autoclaved R36 Lactamin chow (Lactamin), given steriledrinking water ad libitum, and kept under 12-hour light/dark cycles.

The protocols were approved by the Regional Animal Research Eth-ical Board, Stockholm, Sweden, following the proceedings described inthe (European Union) EU legislation (Council Directive 2010/63/EU)and the Institutional Animal Care and Use Committee at The FeinsteinInstitute for Medical Research, Manhasset, NY, USA.

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Colonization of germ-free miceFor colonization, 8- to 10-week-old C57BL/6J germ-free mice receivedfecal matter from the pathogen-free mice through a single gavage andthen were left for 14 days (CONV) before being sacrificed. The controlgroup was gavaged with sterile PBS.

C. tyrobutyricum (DSM 2637) (a contribution of H. Kozakova, De-partment of Immunology and Gnotobiology, Institute of Microbiology,Academy of Sciences of the Czech Republic, Praha, Czech Republic)and B. thetaiotaomicron (BTeta) were cultured in Bryant Burkey brothwith resazurine (Merck), and 108 bacteria were used to colonize eachC57BL/6J germ-free adult male mouse as previously described (19). Four-teen days later, the mice were treated with Evans blue dye to assess theBBB permeability. Another group of germ-free male mice was gavagedfor 3 days with sodium butyrate [1 g/kg body weight (BW) per day]before sacrifice. A group of germ-free male mice and another group ofpathogen-free mice were gavaged with sterile water and used as controls.

Drug treatmentA group of adult male pathogen-free mice receiving a single intra-venous injection of TNFa (100 mg/kg BW) 15 hours before Evans blueperfusion were used as a positive control for a “leaky” BBB (51).

Perfusion with Evans blue dyeEvans blue perfusion was performed as previously described (52).Briefly, anesthetized mice were perfused with PBS followed by Evansblue cocktail. Tissue cryosections were analyzed by fluorescence micros-copy. A detailed description is provided in Supplementary Materials.

In vivo PET imaging of [11C]racloprideScans were performed in a microPET Focus 120 scanner (CTI Con-corde Microsystems). The tracer (maximum volume, 200 ml) was ad-ministered by bolus injection via the tail vein into anesthetized mice.PET data were acquired in full three-dimensional mode, and imageswere reconstructed by standard two-dimensional filtered back projec-tion using a ramp filter. The % SUV is the regional concentration oftissue radioactivity normalized for injected dose and BW. A completedescription is provided in Supplementary Materials.

R4A antibody injection and quantitative analysisGerm- and pathogen-free adult male mice received intravenous injec-tions of R4A dissolved in PBS. Forty-eight hours later, anesthetizedmice were given transcardiac perfusion with 0.9% NaCl followed by4% paraformaldehyde (PFA) (Histolab). Tissue cryosections stainedwith cresyl violet and neurons were sampled from comparable regionsof the anterior dorsal hippocampus (53, 54). A detailed protocol isprovided in Supplementary Materials.

Intravital two-photon laser scanning microscopyTRITC-dextran (155 kD, Sigma) was injected retro-orbitally before im-aging.Two-photon imagingwas performedon aTriMScope II (LaVisionBioTec) equippedwith anOlympus BX51uprightmicroscope fittedwith a20 × 0.95 numerical aperture water immersion objective and a ChameleonUltra-II Tunable,Mode-lockedTi:Sapphire laser (Coherent) tuned to880nmfor excitation of TRITC-dextran. Imaris (Bitplane) was used for three-dimensional image analysis. Volumetric index (VI), a parameter that indi-cates percentage of volumeoccupied bybloodvesselswithin the analyzedZstack, was calculated according to the following formula:VI = total bloodvessel volume within the Z stack/total volume of the Z stack × 100.

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ImmunofluorescenceImmunofluorescence was performed on 50-mm brain coronal cryotomesections blocked with 5% bovine serum albumin (BSA) (Sigma) and0.5% Triton X-100 (Sigma) in PBS, followed by incubation with pri-mary antibody as follows: rat anti-CD13 (BD Pharmingen, 558744),mouse anti-occludin (1:300, Invitrogen, 33-1500), mouse anti–claudin-5(1:300, Invitrogen, 35-2500), and rabbit anti-laminin (1:400, Sigma, L9393).Sections were then incubated with secondary donkey anti-rat Alexa 488,goat anti-mouse Alexa 488, or goat anti-rabbit Alexa 594 (1:500, Invitro-gen) for 1 hour at room temperature. Z stack images were visualized andacquired using a Nikon Eclipse TE300 inverted microscope integratedwith a PerkinElmer UltraVIEW spinning disk confocal system. Imageprocessing was done using Corel Paint Shop Pro Photo XI software.

Protein extraction and Western blot analysisProtein expression was determined in brain lysates as previously de-scribed (19). Primary antibodies (occludin: 1:1000, Invitrogen, 331500;claudin-5: 1:1000, Invitrogen, 352500; and ZO-1: 1:2000, Invitrogen,617300) diluted in 3% BSA in 0.1% Tween 20 (Sigma)/PBS were in-cubated overnight at 4°C with the blot. Protein bands were visualizedby chemiluminescence using Immun-Star WesternC chemilumines-cence kit (Bio-Rad). Protein expression was then quantified usingImageJ software (National Institute of Mental Health).

Transmission electron microscopyA group of adult pathogen- and germ-free mice were transcardially per-fused with 2.5% glutaraldehyde (Sigma)/1% PFA and processed fortransmission electron microscopy. Digital images were taken with a Ve-leta camera (Olympus Soft Imaging Solutions, GmbH). The full method isdescribed in Supplementary Materials.

Statistical analysisStatistical significance was determined using one-way ANOVA withTukey post hoc test for multiple groups, Bonferroni post hoc analysisfor Fig. 1B, or t test when changes were compared between two groups(GraphPad Prism 6). P < 0.05 was considered statistically significantunless otherwise stated. Values were expressed as means ± SEM.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/6/263/263ra158/DC1Material and MethodsFig. S1. Expression of tight junction proteins in the brains of germ- and pathogen-free adultfemale mice.Fig. S2. Electron micrographs showing different tight junction structure (white arrows) in thebrains of germ-free adult mice.Fig. S3. The effect of oral treatment with the bacterial metabolite sodium butyrate or monocolo-nization with C. tyrobutyricum on histone acetylation in extracts of mouse brain frontal cortex.

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Acknowledgments: We thank V. Arulampalam and C. Betsholtz for their valuable comments.We thank A. Samuelsson, J. Aspsäter, and A. A. Bautista Amoyo for technical assistance.Funding: This project was supported by grants from Vetenskapsrådet, EU project “MolecularTargets Open for Regulation by the gut flora—New Avenues for improved Diet to OptimizeEuropean health,” Hjärnfonden, Mérieux Institute, and Singapore Millennium Foundation and

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funding from Lee Kong Chian School of Medicine, Nanyang Technological University to S.P.and grants from the NIH and the Simons Foundation to B.D. We also thank the Wenner-GrenFoundation for postdoctoral funding of V.B.; the Agency for Science, Technology and Researchfor the postdoctoral funding of F.A.; the Higher Education Institute and Qatar University for thePh.D. funding of M.A.-A.; Karolinska Institutet doctoral funding for the Ph.D. scholarship to A.A.;and the Center for Innovative Medicine. Author contributions: V.B., F.A., B.D., and S.P. con-ceived of and designed the project. C.K. and M.A.-A. determined the permeability of the BBB inthe fetal mice; M.T. performed the in vivo PET imaging; V.B., A.A., M.A.-A., and F.A. performedthe Evans blue dye study; V.B. and B.T.V. performed the R4A injection and quantitative anal-ysis; N.B. conducted the two-photon study; A.A. performed the immunofluorescence studies;V.B. performed the Western blot study for tight junction proteins, and F.A. performed theWestern blot data for histone acetylation; K.H. and H.N. performed electron microscopy;and P.K. and M.A.-A. were involved in the animal experiments concerning the role of SCFAs.

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V.B., F.A., M.A.-A., C.K., M.T., B.T.V., N.B., N.L.G., B.D., A.K., and S.P. analyzed the data. V.B. and S.P.wrote the manuscript with significant input from B.G., C.H., N.L.G., C.K., H.H., M.A.-A., and B.D.Competing interests: The authors declare that they have no competing interests.

Submitted 11 June 2014Accepted 31 October 2014Published 19 November 201410.1126/scitranslmed.3009759

Citation: V. Braniste, M. Al-Asmakh, C. Kowal, F. Anuar, A. Abbaspour, M. Tóth, A. Korecka, N. Bakocevic,N. L. Guan, P. Kundu, B. Gulyás, C. Halldin, K. Hultenby, H. Nilsson, H. Hebert, B. T. Volpe, B. Diamond,S. Pettersson, The gut microbiota influences blood-brain barrier permeability in mice. Sci. Transl.Med. 6, 263ra158 (2014).

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